We are continuing to investigate early changes in the synthesis of DNA and its associated proteins during the onset of transformation. We will analyze the synthesis of DNA and proteins binding to DNA during the onset of transformation in Rat-1 cells transformed by avian sarcoma viruses. DNA will be examined by CsCl gradient analysis in order to examine differences in DNA synthesis in the presence and absence of transformation or serum. We are purifying DNA-binding proteins whose synthesis has already been associated with the onset of transformation, in order to test their biological activity in DNA-unwinding and DNA-replication assays in vitro. The relationship of the synthesis of these proteins to transformation and serum-stimulation will be investigated in several cell lines transformed by a variety of oncogenic viruses. The ability of these proteins and cellular protein extracts of transformed cells to influence the replication of DNA in permeabilized cells in vitro will be investigated. The DNA replication in vitro will be analyzed by density gradient centrifugation in neutral and alkaline CsCl. Alterations in the phosphorylation of the DNA-binding proteins will be analyzed during the onset of transformation.